This project investigates how chemical toxins or physical factors alter metabolic processes. NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. Project 1. During the past year, the research focused on a collaborative study with the Shears group (Laboratyr of Signal Transduction) aimed at the characterization of an inositol phosphate metabolizing enzyme. Inositol trisphosphate kinases (IP3Ks) and inositol hexakisphosphate kinases (IP6Ks) each regulate specialized signaling activities by phosphorylating either inositol tri- or hexa-phosphates, respectively. During the past year, we utilized NMR spectroscopy to characterize the products formed by inositol hexaphosphate kinase. The structure of an Entamoeba histolytica hybrid IP6K/IP3K, an enzymatic parallel to a living fossil has been characterized by X-ray crystallography. Through molecular modeling and mutagenesis, we extrapolated our findings to human IP6K2, which retains vestigial IP3K activity. Two structural elements, an alpha-helical pair and a rare, two-turn 3/10 helix, together form a substrate-binding pocket with an open clamshell geometry. InsP6 forms substantial contacts with both structural elements. Relative to InsP6, enzyme-bound InsP3 rotates 55 closer to the alpha-helices, which provide most of the proteins interactions with InsP3. These data reveal the molecular determinants of IP6K activity, and suggest an unusual evolutionary trajectory for a primordial kinase that could have favored efficient bifunctionality, before propagation of separate IP3Ks and IP6Ks. Project 2. Free radical detection with ESR spin trapping relies on the specific addition of the radical to nitrone/nitroso compounds. It also has been proposed that spin traps can react in biological systems to give false-positive results. For nitrone spin traps, the reaction with nucleophiles, first described by Forrester and Hepburn, has been discussed as the most critical source of artifacts. For artifact identification, the ESR preincubation method may be used, which employs isotopically marked spin traps. In collaboration with the Mason group (Laboratory of Toxicology and Pharmacology), we recently investigated the influence of fast sulfite hydroxylamine equilibrium chemistry on the validity of this assay. Using the (faster) aspiration technique, we found that the Forrester-Hepburn mechanism also contributes to DMPO/SO3- adduct formation during ferricyanide-mediated sulfite oxidation, but no evidence for artifactual DMPO/SO3- formation was found if the more potent horseradish peroxidase was used. This provides ESR evidence that the Forrester-Hepburn mechanism can occur under mild conditions, depending on the experimental details. This technique can also be used to test for other artifact mechanisms. We investigated the known ene reaction of DBNBS and tryptophan in more detail. We found that a strong artifact signal is induced by light; however, with atypically long incubations, we found that the artifact is also formed thermally.